The targeting of tumor tissues by antibodies able to selectively recognize antigens overexpressed at the tumor site is the goal of all anti-tumor immunotherapies. Epithelial cell adhesion molecule (EpCAM, swiss prot no. P16422) also known as Tumor-associated calcium signal transducer 1, TROP-1, GA733-2, etc. is a type I trans-membrane glycoprotein expressed at the basolateral membrane on simple epithelia where it is involved in calcium-independent homophilic cell adhesion The extracellular domain consists of one epidermal growth factor like repeat, followed by a thyroglobulin repeat and a cystein poor region while the intracellular domain is 26 amino acids long and there are two binding sites for α-actinin for linkage to the actin cytoskeleton (Winter M J et al. 2003). Cells from major human malignancies strongly overexpress EpCAM as recently confirmed by Went and coworkers (2006) that reported the immunohistochemistry results obtained on a large number of colon, gastric, prostate and lung tumor specimens. EpCAM overexpressing cells tend to segregate from normal cells, correlating with the development of a proliferative and malignant phenotype (Winter et al., 2003). Based on these data, the possibility to target EpCAM for immunotherapy was explored.
A murine IgG2a antibody, Edrecolomab, with some antibody dependent cellular cytotoxicity (ADCC) towards EpCAM positive cells, was approved in Germany for clinical use and employed for the treatment of patients with colorectal or pancreatic carcinoma who had undergone curative surgery. A seven-year follow up outcome of minimal residual disease after edrecolomab treatment in Dukes'C colorectal cancer patients showed a reduced mortality of 32% and a decrease in recurrence rate by 23% (Riethmuller G et al., 1998; Weiner L M et al., 1993). However, the overall results of the clinical study lead to the withdrawn of this drug as monotherapy, due to its limited antitumor efficacy (Punt C J, et al., 2002) and to the promotion of new clinical trials where the antibody is intended in adjuvant settings with other active chemotherapeutic compounds (Frodin J E et al., 2002).
Other antibodies against this target have been used in preclinical or clinical settings. MT-201 (Adecatumumab) is a fully human monoclonal IgG1 antibody with moderate affinity for EpCAM (Naundorf S et al., 2002). Its efficacy was demonstrated in nude mouse xenograft model using the colon cancer cell line HT-29 (Naundorf S et al., 2002). In vitro studies on various tumor cell lines have shown that MT201 mediates target cell lysis by ADCC and complement dependent cytotoxicity (CDC) (Prang N et al., 2005). This antibody is currently under clinical development for the treatment of hormone refractory prostate cancer (Oberneder R et al., 2006).
ING-1 is a high affinity human engineered monoclonal antibody that targets EpCAM positive cells. It has been used in a phase I clinical trial in patients with advanced adenocarcinomas, refractory to standard therapy and the data from this study suggested that antibodies with high affinity to EpCAM, while being more cytotoxic to tumor cells, can also induce rapid pancreatic toxic injury thus, limiting their therapeutic window for systemic administration (De Bono J S et al., 2004). There are diverging opinions on the relevance of antibody affinity for the efficacy of immunotherapy. Velders et al. (1998) presented in vitro data on the impact of antibody affinity and antigen density on ADCC as obtained by comparison of two antibodies having different affinity for EpCAM. Data obtained from this study revealed that the high affinity antibody could mediate cell killing with low antigen expression levels or, at comparable binding levels, with higher efficacy. As heterogeneity of a target antigen expression is a common feature of all tumors, the use of high affinity antibodies could improve clinical results. The possible systemic toxic effects, associated with the therapeutic use of high affinity anti-EpCAM antibodies, might be reduced by pre-targeting strategies which include a chasing step to eliminate, at a given time, the circulating antibody. Alternatively, the use of high affinity anti-EpCAM antibodies might be restricted to loco-regional treatments.
A humanized single chain Fv antibody fragment, NR-LU-10, specific to the EpCAM antigen, genetically engineered as a streptavidin fusion protein has been developed for pre-targeted radioimmunotherapy or radioimmunoguided breast surgery. Preclinical data showed that a single dose of 800 μCi of 90Y-DOTA-biotin administered after NR-LU-10, cured mice with established subcutaneous human small cell lung or colon cancer xenograft (Goshorn S et al., 2001). In a second report by Burak W E Jr et al. (2001) the use of labeled NR-LU-10 Fab was useful in intra-operative probing for revealing tumor localization in 7 out of 10 patients, thus confirming its ability as a targeting agent.
Despite the clinical success of monoclonal antibodies in several pathologies, the immunotherapy of solid tumors still remains unsatisfactory. Pretargeted Antibody Guided RadioImmunoTherapy (PAGRIT™) is a multi-treatment approach allowing restricted and amplified accumulation of the radioisotope in the tumor. The specificity and affinity of the antitumor monoclonal antibody, used as a first step, is fundamental for treatment efficacy.
The Applicant reported exceptionally high and specific accumulation of the ST2146 anti-tenascin monoclonal antibody in both low and high antigen-expressing human xenotransplanted tumors (De Santis et al., Clinical Cancer Research, p. 2191, 1 Apr. 2006).
Based on the teaching of EP 0 496 074, G. Paganelli et al developed this three-step pre-targeting approach for the systemic and loco-regional treatment of tumors (Cremonesi M. et al., Eur. J. Nucl. Med. 26 (2).-110-120, 1999; Paganelli G. et al., Eur. J. Nucl. Med. 26 (4): 348-357, 1999; Paganelli G., et al. Cancer Biother. & Radiopharm. 16 (3): 227-235, 2001).
Other references on the three-step pre-targeting method are WO 94/04702 and U.S. Pat. No. 5,578,287.
The three step pre-targeting treatment is based on intravenous, sequential administration of a biotinylated anti-tenascin monoclonal antibody, streptavidin, and 90Y-labelled biotin with two chasing administrations of avidin and biotinylated albumin before streptavidin and 90Y-labelled biotin, respectively, to reduce non specific background.
In the medical field, there is a need for further and improved anti-EpCAM antibodies useful in cancer diagnosis and therapy, such as for example in the PAGRIT approach.